Benzodiazepine Bromodomain Inhibitor

ABSTRACT

The present invention relates to a benzodiazepine compound, processes for its preparation, pharmaceutical compositions containing such a compound and to its use in therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed as a continuation application of U.S. Ser. No.13/505,039, filed Apr. 30, 2012, which is a National Phase Applicationof International Application No. PCT/EP2010/061518 filed Aug. 6, 2010,which claims priority from GB 0919433.3 filed Nov. 5, 2009 and GB1010509.6 filed Jun. 22, 2010.

FIELD OF THE INVENTION

The present invention relates to a benzodiazepine compound, processesfor its preparation, pharmaceutical compositions containing such acompound and to its use in therapy.

BACKGROUND OF THE INVENTION

The genomes of eukaryotic organisms are highly organised within thenucleus of the cell.

The long strands of duplex DNA are wrapped around an octomer of histoneproteins (most usually comprising two copies of histones H2A, H2B H3 andH4) to form a nuclesome. This basic unit is then further compressed bythe aggregation and folding of nucleosomes to form a highly condensedchromatin structure. A range of different states of condensation arepossible, and the tightness of this structure varies during the cellcycle, being most compact during the process of cell division. Chromatinstructure plays a critical role in regulating gene transcription, whichcannot occur efficiently from highly condensed chromatin. The chromatinstructure is controlled by a series of post translational modificationsto histone proteins, notably histones H3 and H4, and most commonlywithin the histone tails which extend beyond the core nucleosomestructure. These modifications include acetylation, methylation,phosphorylation, ubiquitinylation and SUMOylation. These epigeneticmarks are written and erased by specific enzymes, which place the tagson specific residues within the histone tail, thereby forming anepigenetic code, which is then interpreted by the cell to allow genespecific regulation of chromatin structure and thereby transcription.

Histone acetylation is most usually associated with the activation ofgene transcription, as the modification loosens the interaction of theDNA and the histone octomer by changing the electrostatics. In additionto this physical change, specific proteins bind to acetylated lysineresidues within histones to read the epigenetic code. Bromodomains aresmall (˜110 amino acid) distinct domains within proteins that bind toacetylated lysine resides commonly but not exclusively in the context ofhistones. There is a family of around 50 proteins known to containbromodomains, and they have a range of functions within the cell.

The Bet family of bromodomain containing proteins comprises 4 proteins(BRD2, BRD3, BRD4 and BRD-t) which contain tandem bromodomains capableof binding to two acetylated lysine resides in close proximity,increasing the specificity of the interaction. BRD2 and BRD3 arereported to associate with histones along actively transcribed genes andmay be involved in facilitating transcriptional elongation (Leroy et al,Mol. Cell. 2008 30(1):51-60), while BRD4 appears to be involved in therecruitment of the pTEF-B complex to inducible genes, resulting inphosphorylation of RNA polymerase and increased transcriptional output(Hargreaves et al, Cell, 2009 138(1): 129-145). It has also beenreported that BRD4 and BRD3 fuse with NUT (nuclear protein in testis)forming a novel fusion oncogene, BRD4-NUT, in a highly malignant form ofepithelial neoplasia (French et al. Cancer Research, 2003, 63, 304-307and French et al. Journal of Clinical Oncology, 2004, 22 (20),4135-4139). Data suggests that BRD-NUT fusion proteins contribute tocarcinogensesis (Oncogene, 2008, 27, 2237-2242). BRD-t is uniquelyexpressed in the testes and ovary. All family members have been reportedto have some function in controlling or executing aspects of the cellcycle, and have been shown to remain in complex with chromosomes duringcell division—suggesting a role in the maintenance of epigenetic memory.In addition some viruses make use of these proteins to tether theirgenomes to the host cell chromatin, as part of the process of viralreplication (You et al Cell, 2004 117(3):349-60).

Japanese patent application JP2008-156311 discloses a benzimidazolederivative which is said to be a BRD2 bromodomain binding agent whichhas utility with respect to virus infection/proliferation.

Patent application WO2009/084693A1 discloses a series ofthienotriazolodiazepiene derivatives that are said to inhibit thebinding between an acetylated histone and a bromodomain containg proteinwhich are said to be useful as anti-cancer agents.

A compound has been found which inhibits the binding of bromodomainswith its cognate acetylated proteins, more particularly that inhibitsthe binding of Bet family bromodomains to acetylated lysine residues.Such a compound will hereafter be referred to as a “bromodomaininhibitor”.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a compoundof formula (I) or a salt thereof

In a second aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents or excipients.

In a third aspect of the present invention, there is provided a compoundof formula (I), or a pharmaceutically acceptable salt thereof for use intherapy, in particular in the treatment of diseases or conditions forwhich a bromodomain inhibitor is indicated.

In a fourth aspect of the present invention, there is provided a methodof treating diseases or conditions for which a bromodomain inhibitor isindicated in a subject in need thereof which comprises administering atherapeutically effective amount of compound of formula (I) or apharmaceutically acceptable salt thereof.

In a fifth aspect of the present invention, there is provided the use ofa compound of formula (I), or a pharmaceutically acceptable salt thereofin the manufacture of a medicament for the treatment of diseases orconditions for which a bromodomain inhibitor is indicated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound of formula (I) which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamideor a salt thereof.

It will be appreciated that the present invention covers compounds offormula (I) as the free base and as salts thereof, for example as apharmaceutically acceptable salt thereof.

In one embodiment there is provided a compound which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide.

Because of their potential use in medicine, salts of the compounds offormula (I) are desirably pharmaceutically acceptable. In anotherembodiment there is provided a compound which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamideor a pharmaceutically acceptable salt thereof.

Suitable pharmaceutically acceptable salts can include acid or baseaddition salts. For a review on suitable salts see Berge et al., J.Pharm. Sci., 66:1-19, (1977). Typically, a pharmaceutically acceptablesalt may be readily prepared by using a desired acid or base asappropriate. The resultant salt may precipitate from solution and becollected by filtration or may be recovered by evaporation of thesolvent.

A pharmaceutically acceptable base addition salt can be formed byreaction of a compound of formula (I) with a suitable inorganic ororganic base, (e.g. triethylamine, ethanolamine, triethanolamine,choline, arginine, lysine or histidine), optionally in a suitablesolvent, to give the base addition salt which is usually isolated, forexample, by crystallisation and filtration. Pharmaceutically acceptablebase salts include ammonium salts, alkali metal salts such as those ofsodium and potassium, alkaline earth metal salts such as those ofcalcium and magnesium and salts with organic bases, including salts ofprimary, secondary and tertiary amines, such as isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine andN-methyl-D-glucamine.

A pharmaceutically acceptable acid addition salt can be formed byreaction of a compound of formula (I) with a suitable inorganic ororganic acid (such as hydrobromic, hydrochloric, sulphuric, nitric,phosphoric, succinic, maleic, acetic, propionic, fumaric, citric,tartaric, lactic, benzoic, salicylic, glutamaic, aspartic,p-toluenesulfonic, benzenesulfonic, methanesulfonic, ethanesulfonic,naphthalenesulfonic such as 2-naphthalenesulfonic, or hexanoic acid),optionally in a suitable solvent such as an organic solvent, to give thesalt which is usually isolated, for example, by crystallisation andfiltration. A pharmaceutically acceptable acid addition salt of acompound of formula (I) can comprise or be, for example, a hydrobromide,hydrochloride, sulfate, nitrate, phosphate, succinate, maleate, acetate,propionate, fumarate, citrate, tartrate, lactate, benzoate, salicylate,glutamate, aspartate, p-toluenesulfonate, benzenesulfonate,methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g.2-naphthalenesulfonate) or hexanoate salt.

Other non-pharmaceutically acceptable salts, e.g. formates, oxalates ortrifluoroacetates, may be used, for example, in the isolation of thecompound of formula (I), and are included within the scope of thisinvention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compound of formula (I).

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallized. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or capable of forming hydrogen bonds such aswater, xylene, N-methyl pyrrolidinone, methanol and ethanol may be usedto form solvates. Methods for identification of solvates include, butare not limited to, NMR and microanalysis. Solvates of the compound offormula (I) are within the scope of the invention.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the solvates of the compound of formula (I).

The invention encompasses all prodrugs, of the compound of formula (I)and pharmaceutically acceptable salts thereof, which upon administrationto the recipient are capable of providing (directly or indirectly) acompound of formula (I) or a pharmaceutically acceptable salt thereof,or an active metabolite or residue thereof. Such derivatives arerecognizable to those skilled in the art, without undue experimentation.Nevertheless, reference is made to the teaching of Burger's MedicinalChemistry and Drug Discovery, 5^(th) Edition, Vol 1: Principles andPractice, which is incorporated herein by reference to the extent ofteaching such derivatives.

The compound of formula (I) may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compound of formula(I) may exist as polymorphs, which are included within the scope of thepresent invention. Polymorphic forms of the compound of formula (I) maybe characterized and differentiated using a number of conventionalanalytical techniques, including, but not limited to, X-ray powderdiffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra,differential scanning calorimetry (DSC), thermogravimetric analysis(TGA) and solid state nuclear magnetic resonance (SSNMR).

The compound of formula (I) is an individual isomer isolated such as tobe substantially free of the other isomer (i.e. enantionerically pure)such that less than 10%, particularly less than about 1%, for exampleless than about 0.1% of the other enantiomer is present.

Separation of isomers may be achieved by conventional techniques knownto those skilled in the art, e.g. by fractional crystallisation,chromatography or HPLC.

The compound of formula (I) may exist in one of several tautomericforms. It will be understood that the present invention encompasses alltautomers of the compound of formula (I) whether as individual tautomersor as mixtures thereof.

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, isomers and polymorphic forms of thecompound of formula (I) and salts thereof.

The compound of formula (I) and pharmaceutically acceptable saltsthereof may be made by a variety of methods, including standardchemistry. Illustrative general synthetic methods are set out below andthen the specific compound of formula (I) is prepared in the workingExamples. These processes form further aspects of the present invention.

The compound of formula (I) may be prepared according to reaction scheme1 by reaction of a compound of formula (II) with EtNH₂ in the presenceof HATU or HBTU and DIEA at room temperature. Alternatively compounds offormula (I) may be prepared by reacting the compound of formula (II)with oxalyl chloride followed by addition of EtNH₂ in the presence oftriethylamine.

The compound of formula (II) may be prepared according to reactionScheme 2. Suitable reaction conditions comprise reacting a compound offormula (III) with alkaline hydroxide preferably sodium hydroxide orlithium hydroxide.

wherein R represents C₁₋₆alkyl such as methyl.

Compounds of formula (III), may be prepared according to reaction scheme3 by reacting compounds of formula (IV) with AcOH.

Compounds of formula (IV) may be prepared according to reaction scheme 4by reacting compounds of formula (VI) with hydrazine below 15° C.followed by reaction of the resulting hydrazone (V) with MeCOCl at 0° C.Generally hydrazone (V) is used without further purification and isreacted with MeCOCl at, for example 0° C.

Compounds of formula (VI) in which R is C₁₋₆alkyl (such as methyl) maybe prepared according to reaction scheme 5 from compounds of formula(VII) by treatment with Lawesson's reagent or P₄S₁₀. Suitable reactionconditions comprise reacting compounds of formula (VIII) with P₄S₁₀ in1,2-dichloroethane at, for example 70° C.

Compounds of formula (VII) may be prepared according to reaction scheme6, by reacting compounds of formula (IX) with an organic base such astriethylamine followed by reaction of the resulting amine (VIII) withacetic acid. Generally, amine (VIII) is used without furtherpurification and is reacted with AcOH at, for example 60° C.

Compounds of formula (IX) may be prepared according to reaction scheme7, by reacting compounds of formula (XI) with the acylchloride (X)derived from protected aspartic acid.

Compounds of formula (XI) may be prepared according to proceduresdescribed in Synthesis 1980, 677-688. Acyl chlorides of formula (X) maybe prepared according to procedures described in J. Org. Chem., 1990,55, 3068-3074 and J. Chem. Soc. Perkin Trans. 1, 2001, 1673-1695.

Alternatively the compound of formula (I) may be prepared according toreaction scheme 8.

wherein R represents C₁₋₄alkyl such as methyl.

The compound of formula (IIIA) may be prepared according to reactionscheme 9 by reacting compounds of formula (IVA) with EtNH₂ in thepresence of HATU and DIEA at, for example room temperature.

The compound of formula (IVA) may be prepared according to reactionscheme 10. Suitable reaction conditions comprise reacting compounds offormula (VI) with alkaline hydroxide such as sodium hydroxide.

It will be appreciated by those skilled in the art that it may beadvantageous to protect one or more functional groups of the compoundsdescribed in the above processes. Examples of protecting groups and themeans for their removal can be found in T. W. Greene ‘Protective Groupsin Organic Synthesis’ (4th edition, J. Wiley and Sons, 2006). Suitableamine protecting groups include acyl (e.g. acetyl, carbamate (e.g.2′,2′,2′-trichloroethoxycarbonyl, benzyloxycarbonyl or t-butoxycarbonyl)and arylalkyl (e.g. benzyl), which may be removed by hydrolysis (e.g.using an acid such as hydrochloric acid in dioxane or trifluoroaceticacid in dichloromethane) or reductively (e.g. hydrogenolysis of a benzylor benzyloxycarbonyl group or reductive removal of a2′,2′,2′-trichloroethoxycarbonyl group using zinc in acetic acid) asappropriate. Other suitable amine protecting groups includetrifluoroacetyl (—COCF₃) which may be removed by base catalysedhydrolysis.

It will be appreciated that in any of the routes described above, theprecise order of the synthetic steps by which the various groups andmoieties are introduced into the molecule may be varied. It will bewithin the skill of the practitioner in the art to ensure that groups ormoieties introduced at one stage of the process will not be affected bysubsequent transformations and reactions, and to select the order ofsynthetic steps accordingly.

Certain intermediate compounds described above are believed to be noveland therefore form a yet further aspect of the invention.

The compound of formula (I) and salts thereof is a bromodomaininhibitor, and thus is believed to have potential utility in thetreatment of diseases or conditions for which a bromodomain isindicated.

The present invention thus provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use in therapy. Thecompound of formula (I) or pharmaceutically salt thereof can be for usein the treatment of diseases or conditions for which a bromodomaininhibitor indicated.

In one embodiment there is provided a compound of formula (I) or apharmaceutically acceptable salt thereof for use in the treatment ofdiseases or conditions for which a bromodomain is indicated. In anotherembodiment, there is provided a compound or a pharmaceuticallyacceptable salt thereof for use in the treatment of a chronic autoimmuneand/or inflammatory condition. In a further embodiment, there isprovided a compound or a pharmaceutically acceptable salt thereof foruse in the treatment of cancer, such as midline carcinoma.

In one embodiment there is provided the use of a compound of formula (I)or a pharmaceutically acceptable salt thereof in the manufacture of amedicament for the treatment of diseases or conditions for which abromodomain inhibitor is indicated. In another embodiment, there isprovided the use of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof in the manufacture of a medicament for thetreatment of a chronic autoimmune and/or inflammatory condition. In afurther embodiment, there is provided the use of a compound of formula(I) or a pharmaceutically acceptable salt thereof in the manufacture ofa medicament for the treatment of cancer, such as midline carcinoma.

In one embodiment there is provided a method for treatment of a diseaseor condition, for which a bromodomain inhibitor is indicated, in asubject in need thereof which comprises administering a therapeuticallyeffective amount of compound of formula (I) or a pharmaceuticallyacceptable salt thereof. In another embodiment there is provided amethod for treatment of a chronic autoimmune and/or inflammatorycondition, in a subject in need thereof which comprises administering atherapeutically effective amount of compound of formula (I) or apharmaceutically acceptable salt thereof. In a further embodiment thereis provided a method for treatment of cancer, such as midline carcinoma,in a subject in need thereof which comprises administering atherapeutically effective amount of compound of formula (I) or apharmaceutically acceptable salt thereof.

In one embodiment the subject in need thereof is a mammal, particularlya human.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

Bromodomain inhibitors are believed to be useful in the treatment of avariety of diseases or conditions related to systemic or tissueinflammation, inflammatory responses to infection or hypoxia, cellularactivation and proliferation, lipid metabolism, fibrosis and in theprevention and treatment of viral infections.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof chronic autoimmune and inflammatory conditions such as rheumatoidarthritis, osteoarthritis, acute gout, psoriasis, systemic lupuserythematosus, multiple sclerosis, inflammatory bowel disease (Crohn'sdisease and Ulcerative colitis), asthma, chronic obstructive airwaysdisease, pneumonitis, myocarditis, pericarditis, myositis, eczema,dermatitis, alopecia, vitiligo, bullous skin diseases, nephritis,vasculitis, atherosclerosis, Alzheimer's disease, depression, retinitis,uveitis, scleritis, hepatitis, pancreatitis, primary biliary cirrhosis,sclerosing cholangitis, Addison's disease, hypophysitis, thyroiditis,type I diabetes and acute rejection of transplanted organs.

Bromodomain inhibitors may be useful in the treatment of a wide varietyof acute inflammatory conditions such as acute gout, giant cellarteritis, nephritis including lupus nephritis, vasculitis with organinvolvement such as glomerulonephritis, vasculitis including giant cellarteritis, Wegener's granulomatosis, Polyarteritis nodosa, Behcet'sdisease, Kawasaki disease, Takayasu's Arteritis and acute rejection oftransplanted organs.

Bromodomain inhibitors may be useful in the prevention or treatment ofdiseases or conditions which involve inflammatory responses toinfections with bacteria, viruses, fungi, parasites or their toxins,such as sepsis, sepsis syndrome, septic shock, endotoxaemia, systemicinflammatory response syndrome (SIRS), multi-organ dysfunction syndrome,toxic shock syndrome, acute lung injury, ARDS (adult respiratorydistress syndrome), acute renal failure, fulminant hepatitis, burns,acute pancreatitis, post-surgical syndromes, sarcoidosis, Herxheimerreactions, encephalitis, myelitis, meningitis, malaria, SIRS associatedwith viral infections such as influenza, herpes zoster, herpes simplexand coronavirus.

Bromodomain inhibitors may be useful in the prevention or treatment ofconditions associated with ischaemia-reperfusion injury such asmyocardial infarction, cerebro-vascular ischaemia (stroke), acutecoronary syndromes, renal reperfusion injury, organ transplantation,coronary artery bypass grafting, cardio-pulmonary bypass procedures andpulmonary, renal, hepatic, gastro-intestinal or peripheral limbembolism.

Bromodomain inhibitors may be useful in the treatment of disorders oflipid metabolism via the regulation of APO-A1 such ashypercholesterolemia, atherosclerosis and Alzheimer's disease.

Bromodomain inhibitors may be useful in the treatment of fibroticconditions such as idiopathic pulmonary fibrosis, renal fibrosis,post-operative stricture, keloid formation, scleroderma and cardiacfibrosis.

Bromodomain inhibitors may be useful in the prevention and treatment ofviral infections such as herpes virus, human papilloma virus,adenovirus, poxvirus and other DNA viruses.

Bromodomain inhibitors may be useful in the treatment of cancer,including hematological (such as leukaemia), epithelial including lung,breast and colon carcinomas, midline carcinomas, mesenchymal, hepatic,renal and neurological tumours.

Bromodomain inhibitors may be useful in the treatment of treatment ofophthamological indications such as dry eye.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from diseases associated withsystemic inflammatory response syndrome, such as sepsis, burns,pancreatitis, major trauma, haemorrhage and ischaemia. In thisembodiment the bromodomain inhibitor would be administered at the pointof diagnosis to reduce the incidence of: SIRS, the onset of shock,multi-organ dysfunction syndrome, which includes the onset of acute lunginjury, ARDS, acute renal, hepatic, cardiac and gastro-intestinal injuryand mortality. In another embodiment the bromodomain inhibitor would beadministered prior to surgical or other procedures associated with ahigh risk of sepsis, haemorrhage, extensive tissue damage, SIRS or MODS.In a particular embodiment the disease or condition for which abromodomain inhibitor is indicated is sepsis, sepsis syndrome, septicshock and/or endotoxaemia. In another embodiment, the bromodomaininhibitor is indicated for the treatment of acute or chronicpancreatitis. In another embodiment the bromodomain is indicated for thetreatment of burns.

In one embodiment the disease or condition for which a bromodomaininhibitor is indicated is selected from herpes simplex infections andreactivations, cold sores, herpes zoster infections and reactivations,chickenpox, shingles, human papilloma virus, cervical neoplasia,adenovirus infections, including acute respiratory disease, and poxvirusinfections such as cowpox and smallpox and African swine fever virus. Inone particular embodiment a bromodomain inhibitor is indicated for thetreatment of Human papilloma virus infections of skin or cervicalepithelia.

The term “diseases or conditions for which a bromodomain inhibitor isindicated”, is intended to include any or all of the above diseasestates.

While it is possible that for use in therapy, a compound of formula (I)as well as pharmaceutically acceptable salts thereof may be administeredas the raw chemical, it is common to present the active ingredient as apharmaceutical composition.

The present invention therefore provides in a further aspect apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents or excipients.

Thus there is provided a pharmaceutical composition for the treatment ofdiseases or conditions in which a bromodomain inhibitor is indicatedcomprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof.

The carrier(s), diluent(s) or excipient(s) used in such pharmaceuticalcompositions must be acceptable in the sense of being compatible withthe other ingredients of the composition and not deleterious to therecipient thereof. In accordance with another aspect of the inventionthere is also provided a process for the preparation of a pharmaceuticalcomposition including admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable carriers, diluents or excipients. Thepharmaceutical composition can be for use in the treatment of any of theconditions described herein.

Since the compound of formula (I) and pharmaceutically acceptable saltsthereof are intended for use in pharmaceutical compositions it will bereadily understood that they are each preferably provided insubstantially pure form, for example, at least 60% pure, more suitablyat least 75% pure and preferably at least 85% pure, especially at least98% pure (% in a weight for weight basis).

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Preferred unit dosage compositions are those containing a daily dose orsub-dose, or an appropriate fraction thereof, of an active ingredient.Such unit doses may therefore be administered more than once a day.Preferred unit dosage compositions are those containing a daily dose orsub-dose (for administration more than once a day), as herein aboverecited, or an appropriate fraction thereof, of an active ingredient.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, inhaled, intranasal, topical (including buccal,sublingual or transdermal), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) route. Suchcompositions may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

In one embodiment the pharmaceutical composition is adapted for oraladministration.

In one embodiment the pharmaceutical composition is adapted forparenteral administration, particularly intravenous administration.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders suitable for incorporating intotablets or capsules may be prepared by reducing the compound to asuitable fine size (e.g. by micronisation) and mixing with a similarlyprepared pharmaceutical carrier such as an edible carbohydrate, as, forexample, starch or mannitol. Flavoring, preservative, dispersing andcoloring agent can also be present.

Capsules may be made by preparing a powder mixture, as described above,and filling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, glidants,lubricants, sweetening agents, flavours, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like. Tablets are formulated, for example, bypreparing a powder mixture, granulating or slugging, adding a lubricantand disintegrant and pressing into tablets. A powder mixture is preparedby mixing the compound, suitably comminuted, with a diluent or base asdescribed above, and optionally, with a binder such ascarboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone,a solution retardant such as paraffin, a resorption accelerator such asa quaternary salt and/or an absorption agent such as bentonite, kaolinor dicalcium phosphate. The powder mixture can be granulated by wettingwith a binder such as syrup, starch paste, acadia mucilage or solutionsof cellulosic or polymeric materials and forcing through a screen. As analternative to granulating, the powder mixture can be run through thetablet machine and the result is imperfectly formed slugs broken intogranules. The granules can be lubricated to prevent sticking to thetablet forming dies by means of the addition of stearic acid, a stearatesalt, talc or mineral oil. The lubricated mixture is then compressedinto tablets. The compounds of the present invention can also becombined with a free flowing inert carrier and compressed into tabletsdirectly without going through the granulating or slugging steps. Aclear or opaque protective coating consisting of a sealing coat ofshellac, a coating of sugar or polymeric material and a polish coatingof wax can be provided. Dyestuffs can be added to these coatings todistinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The formulation can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Dosage forms for nasal or inhaled administration may conveniently beformulated as aerosols, solutions, suspensions, gels or dry powders.

For compositions suitable and/or adapted for inhaled administration, itis preferred that the compound of formula (I) or pharmaceuticallyacceptable salt thereof is in a particle-size-reduced form e.g. obtainedby micronisation. The preferable particle size of the size-reduced (e.g.micronised) compound or salt is defined by a D50 value of about 0.5 toabout 10 microns (for example as measured using laser diffraction).

Aerosol formulations, e.g. for inhaled administration, can comprise asolution or fine suspension of the active substance in apharmaceutically acceptable aqueous or non-aqueous solvent. Aerosolformulations can be presented in single or multidose quantities insterile form in a sealed container, which can take the form of acartridge or refill for use with an atomising device or inhaler.Alternatively the sealed container may be a unitary dispensing devicesuch as a single dose nasal inhaler or an aerosol dispenser fitted witha metering valve (metered dose inhaler) which is intended for disposalonce the contents of the container have been exhausted.

Where the dosage form comprises an aerosol dispenser, it preferablycontains a suitable propellant under pressure such as compressed air,carbon dioxide or an organic propellant such as a hydrofluorocarbon(HFC). Suitable HFC propellants include 1,1,1,2,3,3,3-heptafluoropropaneand 1,1,1,2-tetrafluoroethane. The aerosol dosage forms can also takethe form of a pump-atomiser. The pressurised aerosol may contain asolution or a suspension of the active compound. This may require theincorporation of additional excipients e.g. co-solvents and/orsurfactants to improve the dispersion characteristics and homogeneity ofsuspension formulations. Solution formulations may also require theaddition of co-solvents such as ethanol.

For pharmaceutical compositions suitable and/or adapted for inhaledadministration, the pharmaceutical composition may be a dry powderinhalable composition. Such a composition can comprise a powder basesuch as lactose, glucose, trehalose, mannitol or starch, the compound offormula (I) or a pharmaceutically acceptable salt thereof (preferably inparticle-size-reduced form, e.g. in micronised form), and optionally aperformance modifier such as L-leucine or another amino acid and/ormetals salts of stearic acid such as magnesium or calcium stearate.Preferably, the dry powder inhalable composition comprises a dry powderblend of lactose e.g. lactose monohydrate and the compound of formula(I) or a pharmaceutically acceptable salt thereof. Such compositions canbe administered to the patient using a suitable device such as theDISKUS® device, marketed by GlaxoSmithKline which is for exampledescribed in GB 2242134 A.

The compound of formula (I) and pharmaceutically acceptable saltsthereof may be formulated as a fluid formulation for delivery from afluid dispenser, for example a fluid dispenser having a dispensingnozzle or dispensing orifice through which a metered dose of the fluidformulation is dispensed upon the application of a user-applied force toa pump mechanism of the fluid dispenser. Such fluid dispensers aregenerally provided with a reservoir of multiple metered doses of thefluid formulation, the doses being dispensable upon sequential pumpactuations. The dispensing nozzle or orifice may be configured forinsertion into the nostrils of the user for spray dispensing of thefluid formulation into the nasal cavity. A fluid dispenser of theaforementioned type is described and illustrated in WO2005/044354 A1.

A therapeutically effective amount of a compound of formula (I) orpharmaceutically acceptable salt thereof will depend upon a number offactors including, for example, the age and weight of the animal, theprecise condition requiring treatment and its severity, the nature ofthe formulation, and the route of administration, and will ultimately beat the discretion of the attendant physician or veterinarian. In thepharmaceutical composition, each dosage unit for oral or parenteraladministration preferably contains from 0.01 to 3000 mg, more preferably0.5 to 1000 mg, of a compound of formula (I) or pharmaceuticallyacceptable salt thereof calculated as the free base. Each dosage unitfor nasal or inhaled administration preferably contains from 0.001 to 50mg, more preferably 0.01 to 5 mg, of a compound of the formula (I) or apharmaceutically acceptable salt thereof, calculated as the free base.

The compound of formula (I) and pharmaceutically acceptable saltsthereof can be administered in a daily dose (for an adult patient) of,for example, an oral or parenteral dose of 0.01 mg to 3000 mg per day or0.5 to 1000 mg per day, or a nasal or inhaled dose of 0.001 to 50 mg perday or 0.01 to 5 mg per day, of the compound of formula (I) or apharmaceutically acceptable salt thereof, calculated as the free base.This amount may be given in a single dose per day or more usually in anumber (such as two, three, four, five or six) of sub-doses per day suchthat the total daily dose is the same. An effective amount of apharmaceutically acceptable salt thereof, may be determined as aproportion of the effective amount of the compound of formula (I) perse.

Thus there is provided a pharmaceutical composition comprising a) 0.01to 3000 mg of a compound of formula (I) or a pharmaceutically acceptablesalt thereof, and (b) 0.1 to 2 g of one or more pharamceuticallyacceptable carriers, diluents and/or excipients.

The compound of formula (I) and pharmaceutically acceptable saltsthereof may be employed alone or in combination with other therapeuticagents. Combination therapies according to the present invention thuscomprise the administration of at least one compound of formula (I) or apharmaceutically acceptable salt thereof, and the use of at least oneother pharmaceutically active agent. Preferably, combination therapiesaccording to the present invention comprise the administration of atleast one compound of formula (I) or a pharmaceutically acceptable saltthereof, and at least one other pharmaceutically active agent. Thecompound of formula (I) and pharmaceutically acceptable salts thereof,and the other pharmaceutically active agent(s) may be administeredtogether in a single pharmaceutical composition or separately and, whenadministered separately this may occur simultaneously or sequentially inany order. The amounts of the compound of formula (I) andpharmaceutically acceptable salts thereof, and the otherpharmaceutically active agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect. Thus in a further aspect, there is provided acombination comprising a compound of formula (I) or pharmaceuticallyacceptable salt thereof and at least one other pharmaceutically activeagent. In one embodiment there is provided a combination pharmaceuticalproduct comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof together with one or more other therapeuticallyactive agents.

Thus in one aspect, the compound of formula (I) and pharmaceuticalcompositions according to the invention may be used in combination withor include one or more other therapeutic agents, for example selectedfrom antibiotics, anti-virals, glucocorticosteroids, muscarinicantagonists and beta-2 agonists.

It will be appreciated that when the compound of formula (I) andpharmaceutically acceptable salt thereof the are administered incombination with other therapeutic agents normally administered by theinhaled, intravenous, oral or intranasal route, that the resultantpharmaceutical composition may be administered by the same routes.Alternatively the individual components of the composition may beadministered by different routes.

One embodiment of the invention encompasses combinations comprising oneor two other therapeutic agents.

It will be clear to a person skilled in the art that, where appropriate,the other therapeutic ingredient(s) may be used in the form of salts,for example as alkali metal or amine salts or as acid addition salts, orprodrugs, or as esters, for example lower alkyl esters, or as solvates,for example hydrates, to optimise the activity and/or stability and/orphysical characteristics, such as solubility, of the therapeuticingredient. It will be clear also that, where appropriate, thetherapeutic ingredients may be used in optically pure form.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with apharmaceutically acceptable diluent or carrier represent a furtheraspect of the invention.

The compound of formula (I) may be prepared by the methods describedbelow or by similar methods. Thus the following Intermediates andExamples serve to illustrate the preparation of the compound of formula(I) and is not to be considered as limiting the scope of the inventionin any way.

General Experimental Details

All temperatures referred to are in ° C.

Abbreviations

-   -   TLC—thin layer chromatography    -   AcOH—acetic acid    -   AcCl—acetyl chloride    -   PPTS—pyridinium p-toluenesulfonate    -   DCM—dichloromethane    -   1,2-DCE—1,2-dichloroethane    -   DIC—Diisopropylcarbodiimide    -   DIEA—N,N-diisopropylethylamine    -   DMF—N,N-dimethylformamide    -   DMAP—4-dimethylaminopyridine    -   Fmoc—9H-fluoren-9-ylmethyl)oxy]carbonyl    -   HATU—O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HBTU—O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   Et₂O—diethyl ether    -   EtOAc—ethyl acetate    -   i-Pr₂O—di-isopropyl ether    -   Config.—absolute configuration    -   Lawesson's—2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulphide        Reagent    -   MeCN—acetonitrile    -   MeOH—methanol    -   Rt—retention time    -   THF—tetrahydrofuran    -   RT—room temperature    -   Pd/C—palladium on carbon

LC/MS refers to analyses by analytical HPLC which were conducted on twokinds of apparatus:

-   -   a) On a Supelcosil LCABZ+PLUS column (3 μm, 3.3 cm×4.6 mm ID)        eluting with 0.1% HCO₂H and 0.01 M ammonium acetate in water        (solvent A), and 95% acetonitrile and 0.05% HCO₂H in water        (solvent B), using the following elution gradient 0-0.7 minutes        0% B, 0.7-4.2 minutes 0→100% B, 4.2-5.3 minutes 100% B, 5.3-5.5        minutes 100→0% B at a flow rate of 3 mL/minute. The mass spectra        (MS) were recorded on a Fisons VG Platform mass spectrometer        using electrospray positive ionisation [(ES+ve to give [M+H]⁺        and [M+NH₄]⁺ molecular ions] or electrospray negative ionisation        [(ES−ve to give [M−H]− molecular ion] modes. Analytical data        from this apparatus are given with the following format : [M+H]⁺        or [M−H]⁻.    -   b) On a Chromolith Performance RP 18 column (100×4.6 mm id)        eluting with 0.01M ammonium acetate in water (solvent A) and        100% acetonitrile (solvent B), using the following elution        gradient 0-4 minutes 0→100% B, 4-5 minutes 100% B at a flow rate        of 5 mL/minute. The mass spectra (MS) were recorded on a        micromass Platform-LC mass spectrometer using atmospheric        pressure chemical positive ionisation [AP+ve to give MH⁺        molecular ions] or atmospheric pressure chemical negative        ionisation [AP−ve to give (M−H)⁻ molecular ions] modes.        Analytical data from this apparatus are given with the following        format: [M+H]+ or [M−H]− preceded by the acronym APCI to specify        between both mass spectrometry analyses sources.

LC/HRMS: Analytical HPLC was conducted on a Uptisphere-hsc column (3 μm33×3 mm id) eluting with 0.01M ammonium acetate in water (solvent A) and100% acetonitrile (solvent B), using the following elution gradient0-0.5 minutes 5% B, 0.5-3.75 minutes 5→100% B, 3.75-4.5 100% B, 4.5-5100→5% B, 5-5.5 5% B at a flow rate of 1.3 mL/minute. The mass spectra(MS) were recorded on a micromass LCT mass spectrometer usingelectrospray positive ionisation [ES+ve to give MH⁺ molecular ions] orelectrospray negative ionisation [ES−ve to give (M−H)− molecular ions]modes.

Mass directed auto-prep HPLC refers to the method where the material waspurified by high performance liquid chromatography on a HPLCABZ+ 5 μmcolumn (5 cm×10 mm i.d.) with 0.1% HCO₂H in water and 95% MeCN, 5% water(0.5% HCO₂H) utilising the following gradient elution conditions: 0-1.0minutes 5% B, 1.0-8.0 minutes 5→30% B, 8.0-8.9 minutes 30% B, 8.9-9.0minutes 30→95% B, 9.0-9.9 minutes 95% B, 9.9-10 minutes 95→0% B at aflow rate of 8 mL/minute. The Gilson 202-fraction collector wastriggered by a VG Platform Mass Spectrometer on detecting the mass ofinterest.

Proton NMR (¹H NMR) spectra were recorded at ambient temperature on aBruker Avance 300 DPX spectrometer using solvant as internal standardand proton chemical shifts are expressed in ppm in the indicatedsolvent. The following abbreviations are used for multiplicity of NMRsignals: s=singlet, d=doublet, t=triplet, q=quadruplet, dd=doubledoublet, m=multiplet.

TLC (thin layer chromatography) refers to the use of TLC plates sold byMerck coated with silica gel 60 F254.

EXAMPLE 12-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide

To a solution of[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid (for a preparation see Intermediate 1)(16.0 g, 40 mmol) in THF atRT was added DIEA (14 mL, 80 mmol) followed by HATU (30.4 g, 80 mmol).The reaction mixture was stirred for 3 h at this temperature andethylamine (40 mL, 2M in THF, 80 mmol) was added. The mixture wasstirred for 48 h before being concentrated under reduced pressure. Thecrude material was suspended in water and extracted with DCM. Theorganic layer was dried over Na₂SO₄, filtered and concentrated in vacuo.The crude solid was purified by chromatography on SiO₂ (DCM/MeOH 95/5)and the resulting solid recrystallised in MeCN. The solid was thendissolved in DCM and precipited with i-Pr₂O to give the title compound(8 g, 47% yield) as a white solid.

R_(f)=0.48 (DCM/MeOH: 90/10). Mp>140° C. (becomes gummy). ¹H NMR (300MHz, CDCl₃) δ 7.53-7.47 (m, 2H), 7.39 (d, J=8.9 Hz, 1H), 7.37-7.31 (m,2H), 7.20 (dd, J=2.9 and 8.9 Hz, 1H), 6.86 (d, J=2.9 Hz, 1H), 6.40 (m,1H), 4.62 (m, 1H), 3.80 (s, 3H), 3.51 (dd, J=7.3 and 14.1 Hz, 1H),3.46-3.21 (m, 3H), 2.62 (s, 3H), 1.19 (t, J=7.3 Hz, 3H). LC/MS: m/z 424[M(³⁵Cl)+H]⁺, Rt 2.33 min.

Intermediate 1:[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid

To a solution ofmethyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate(for a preparation see Intermediate 2)(28 g, 68 mmol) in THF (450 mL) atRT was added 1N NaOH (136 mL, 136 mmol). The reaction mixture wasstirred at this temperature for 5 h before being cooled down andquenched with 1N HCl (136 mL). THF was removed under reduced pressureand the aqueous layer was extracted with DCM. The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure. The crude solid was recrystallised in CH₃CN to give the titlecompound (23.9 g, 89% yield) as a pale yellow powder. ¹H NMR (300 MHz,CDCl₃) δ 7.55-7.48 (m, 2H), 7.41 (d, J=8.9 Hz, 1H), 7.38-7.31 (m, 2H),7.22 (dd, J=2.9 and 8.9 Hz, 1H), 6.90 (d, J=2.9 Hz, 1H), 4.59 (dd, J=6.9and 6.9 Hz, 1H), 3.81 (s, 3H), 3.70 (dd, J=6.9 and 25.7 Hz, 1H), 3.61(dd, J=6.9 and 25.7 Hz, 1H), 2.63 (s, 3H). LC/MS: m/z 397 [M(³⁵Cl)+H]⁺,Rt 2.11 min.

Intermediate 2:Methyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate

To crudemethyl[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Intermediate 3) (34 g, 79 mmol) was suspended inTHF (200 mL) and AcOH (200 mL) was added at RT. The reaction mixture wasstirred at this temperature overnight before being concentrated todryness. The residue was suspended in saturated NaHCO₃ and extractedwith DCM. The organic layer was dried over Na₂SO₄, filtered andconcentrated in vacuo. The crude solid was purified by chromatography onSiO₂ (DCM/MeOH: 90/10) to give the title compound (28 g, 86% yield) as ayellow powder.

¹H NMR (300 MHz, CDCl₃) δ 7.54-7.47 (m, 2H), 7.40 (d, J=8.8 Hz, 1H),7.37-7.31 (m, 2H), 7.22 (dd, J=2.8 and 8.8 Hz, 1H), 6.89 (d, J=2.8 Hz,1H), 4.61 (dd, J=6.4 and 7.8 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.66(dd, J=7.8 and 16.9 Hz, 1H), 3.60 (dd, J=6.4 and 16.9 Hz, 1H), 2.62 (s,3H). LC/MS m/z 411 [M(³⁵Cl)+H]⁺, Rt 2.88 min.

Intermediate 3:Methyl[(3S)-2-[2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate

To a suspension ofmethyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Intermediate 4)(30.2 g, 77.7 mmol) in THF (800mL) at 0° C. was added hydrazine monohydrate (11.3 mL, 233 mmol)dropwise. The reaction mixture was stirred for 4 h between 0° C. and 15°C. before being cooled at 0° C. Et₃N (32.4 mL, 230 mmol) was then addedslowly and AcCl (16.3 mL, 230 mmol) was added dropwise. The mixture wasallowed to warm to RT and stir for 1 h then quenched with water andconcentrated under reduced pressure. The resulting aqueous layer wasthen extracted with DCM and the organic layer was dried over Na₂SO₄,filtered and concentrated in vacuo to give the crude title compound (34g, 100% yield) which was used without further purification. LC/MS: m/z429 [M(³⁵Cl)+H]⁺, Rt 2.83 min.

Intermediate 4:Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

A suspension of P₄S₁₀ (85.8 g, 190 mmol) and Na₂CO₃ (20.5 g, 190 mmol)in 1,2-DCE (1.5 L) at RT was stirred for 1 h beforemethyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Intermediate 5) (40 g, 107 mmol) was added. Theresulting mixture was stirred at 65° C. for 4 h before being cooled andfiltered. The solid was washed with DCM and the filtrate washed withsat. NaHCO₃. The organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The title compound was precipitatedfrom a DCM/i-Pr₂O mixture and filtered. The filtrate was thenconcentrated and purified by flash chromatography (DCM/MeOH: 98/2) toafford another batch of product. The title compound was obtainedcombining the two fractions (30.2 g, 73%) as a yellow powder. LC/MS: m/z389 [M(³⁵Cl)+H]⁺, Rt 3.29 min.

Intermediate 5:Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

To a solution of the crude methylN¹-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N²-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate(for a preparation see Intermediate 6) (assumed 0.2 mol) in DCM (500 mL)was added Et₃N (500 mL, 3.65 mol) and the resulting mixture was refluxedfor 24 h before being concentrated. The resulting crude amine wasdissolved in 1,2-DCE (1.5 L) and AcOH (104 mL, 1.8 mol) was addedcarefully. The reaction mixture was then stirred at 60° C. for 2 hbefore being concentrated in vacuo and dissolved in DCM. The organiclayer was washed with 1N HCl and the aqueous layer was extracted withDCM (×3). The combined organic layers were washed twice with water, andbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The crude solid was recrystallised in MeCN leading to thetitle compound (51 g) as a pale yellow solid. The filtrate could beconcentrated and recrystallised in MeCN to give another 10 g ofIntermediate 9 (total: 61 g, 69% yield based on recovered Intermediate12). R_(f)=0.34 (DCM/MeOH: 95/5). LC/MS m/z 373 [M(³⁵Cl)+H]⁺, Rt 2.76min.

Intermediate 6: MethylN¹-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N²-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate

A mixture of MethylN-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-aspartyl chloride (preparedfrom J. Org. Chem. 1990, 55, 3068-3074 and J. Chem. Soc. Perkin Trans. 12001, 1673-1695) (221 g, 0.57 mol) and[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone (for apreparation see Intermediate 7) (133 g, 0.5 mol) in CHCl₃ (410 mL) wasstirred at 60° C. for 1.5 h before being cooled and concentrated underreduced pressure and used without further purification. LC/MS: m/z 613[M(³⁵Cl)+H]⁺, Rt=3.89 min.

Intermediate 7: [2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone

To a solution of 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one (for apreparation see Intermediate 8) (40.0 g, 0.21 mol) in a toluene (560mL)/ether (200 mL) mixture at 0° C. was added dropwise a solution of4-chlorophenylmagnesium bromide (170 mL, 1M in Et₂O, 0.17 mol). Thereaction mixture was allowed to warm to RT and stirred for 1 h beforebeing quenched with 1N HCl. The aqueous layer was extracted with EtOAc(3×) and the combined organics were washed with brine, dried overNa₂SO₄, filtered and concentrated under reduced pressure. The crudecompound was then dissolved in EtOH (400 mL) and 6N HCl (160 mL) wasadded. The reaction mixture was refluxed for 2 h before beingconcentrated under reduced pressure. The resulting solid was filteredand washed twice with ether before being suspended in EtOAc andneutralised with 1N NaOH. The aqueous layer was extracted with EtOAc(3×) and the combined organics were washed with brine, dried overNa₂SO₄, filtered and concentrated under reduced pressure. The titlecompound was obtained as a yellow solid (39 g, 88% yield) which was usedwithout further purification.

Intermediate 8 : 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one

A solution of 5-methoxyanthranilic acid (7.8 g, 46.5 mmol) was refluxedin acetic anhydride (60 mL) for 2 h15 before being cooled andconcentrated under reduced pressure. The crude residue was thenconcentrated twice in the presence of toluene before being filtered andwashed with ether to yield to the title compound (6.8 g, 77% yield) as abeige solid; LC/MS: m/z 192 [M+H]⁺, Rt 1.69 min.

Preparation of Reference Compound for Use in Biological Assays

Experimental details of LC-MS methods A and B as referred to herein areas follows:

LC/MS (Method A) was conducted on a Supelcosil LCABZ+PLUS column (3 μm,3.3 cm×4.6 mm ID) eluting with 0.1% HCO₂H and 0.01 M ammonium acetate inwater (solvent A), and 95% acetonitrile and 0.05% HCO₂H in water(solvent B), using the following elution gradient 0-0.7 minutes 0% B,0.7-4.2 minutes 0→100% B, 4.2-5.3 minutes 100% B, 5.3-5.5 minutes 100→0%B at a flow rate of 3 mL/minute. The mass spectra (MS) were recorded ona Fisons VG Platform mass spectrometer using electrospray positiveionisation [(ES+ve to give [M+H]⁺ and [M+NH₄]⁺ molecular ions] orelectrospray negative ionisation [(ES−ve to give [M−H]− molecular ion]modes. Analytical data from this apparatus are given with the followingformat: [M+H]⁺ or [M−H]⁻.

LC/MS (Method B) was conducted on an Sunfire C18 column (30 mm×4.6 mmi.d. 3.5 μm packing diameter) at 30 degrees centigrade, eluting with0.1% v/v solution of Trifluoroacetic Acid in Water (Solvent A) and 0.1%v/v solution of Trifluoroacetic Acid in Acetonitrile (Solvent B) usingthe following elution gradient 0-0.1 min 3% B, 0.1-4.2 min 3-100% B,4.2-4.8 min 100% B, 4.8-4.9 min 100-3% B, 4.9-5.0min 3% B at a flow rateof 3 ml/min. The UV detection was an averaged signal from wavelength of210 nm to 350 nm and mass spectra were recorded on a mass spectrometerusing positive electrospray ionization. Ionisation data was rounded tothe nearest integer.

LC/HRMS: Analytical HPLC was conducted on a Uptisphere-hsc column (3 μm33×3 mm id) eluting with 0.01M ammonium acetate in water (solvent A) and100% acetonitrile (solvent B), using the following elution gradient0-0.5 minutes 5% B, 0.5-3.75 minutes 5→100% B, 3.75-4.5 100% B, 4.5-5100→5% B, 5-5.5 5% B at a flow rate of 1.3 mL/minute. The mass spectra(MS) were recorded on a micromass LCT mass spectrometer usingelectrospray positive ionisation [ES+ve to give MH⁺ molecular ions] orelectrospray negative ionisation [ES−ve to give (M−H)− molecular ions]modes.

TLC (thin layer chromatography) refers to the use of TLC plates sold byMerck coated with silica gel 60 F254.

Silica chromatography techniques include either automated (Flashmasteror Biotage SP4) techniques or manual chromatography on pre-packedcartridges (SPE) or manually-packed flash columns.

Reference Compound A: 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one

A solution of 5-methoxyanthranilic acid (Lancaster) (41.8 g, 0.25 mol)was refluxed in acetic anhydride (230 mL) for 3.5 h before beingconcentrated under reduced pressure. The crude compound was thenconcentrated twice in the presence of toluene before being filtered andwashed twice with ether to yield to the title compound (33.7 g, 71%yield) as a brown solid; LC/MS (Method A): m/z 192 [M+H]⁺, Rt 1.69 min.

Reference Compound B:[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone

To a solution of 2-methyl-6-(methyloxy)-4H-3,1-benzoxazin-4-one (for apreparation see Reference compound A) (40.0 g, 0.21 mol) in atoluene/ether (2/1) mixture (760 mL) at 0° C. was added dropwise asolution of 4-chlorophenylmagnesium bromide (170 mL, 1M in Et₂O, 0.17mol). The reaction mixture was allowed to warm to room temperature andstirred for 1 h before being quenched with 1N HCl (200 mL). The aqueouslayer was extracted with EtOAc (3×150 mL) and the combined organics werewashed with brine (100 mL), dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The crude compound was then dissolved in EtOH(400 mL) and 6N HCl (160 mL) was added. The reaction mixture wasrefluxed for 2 h before being concentrated to one-third in volume. Theresulting solid was filtered and washed twice with ether before beingsuspended in EtOAc and neutralised with 1N NaOH. The aqueous layer wasextracted with EtOAc (3×150 mL) and the combined organics were washedwith brine (150 mL), dried over Na₂SO₄, filtered and concentrated underreduced pressure. The title compound was obtained as a yellow solid (39g, 88% yield); LC/MS (Method A): m/z 262 [M+H]+, Rt 2.57 min.

Reference Compound C: MethylN¹-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N²-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate

Methyl N-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-aspartyl chloride(Int. J. Peptide Protein Res. 1992, 40, 13-18) (93 g, 0.24 mol) wasdissolved in CHCl₃ (270 mL) and[2-amino-5-(methyloxy)phenyl](4-chlorophenyl)methanone (for apreparation see Reference compound B) (53 g, 0.2 mol) was added. Theresulting mixture was stirred at 60° C. for 1 h before being cooled andconcentrated at 60% in volume. Ether was added at 0° C. and theresulting precipitate was filtered and discarded. The filtrate wasconcentrated under reduced pressure and used without furtherpurification.

Reference Compound D:Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

To a solution of MethylN1-[2-[(4-chlorophenyl)carbonyl]-4-(methyloxy)phenyl]-N2-{[(9H-fluoren-9-ylmethyl)oxy]carbonyl}-L-α-asparaginate(for a preparation see Reference compound C) (assumed 0.2 mol) in DCM(500 mL) was added Et₃N (500 mL, 3.65 mol) and the resulting mixture wasrefluxed for 24 h before being concentrated. The resulting crude aminewas dissolved in 1,2-DCE (1.5 L) and AcOH (104 mL, 1.8 mol) was addedcarefully. The reaction mixture was then stirred at 60° C. for 2 hbefore being concentrated in vacuo and dissolved in DCM. The organiclayer was washed with 1N HCl and the aqueous layer was extracted withDCM (×3). The combined organic layers were washed twice with water, andbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. The crude solid was recrystallised in MeCN leading to thetitle compound (51 g) as a pale yellow solid. The filtrate could beconcentrated and recrystallised in MeCN to give to another 10 g of thedesired product R_(f)=0.34 (DCM/MeOH: 95/5).

HRMS (M+H)⁺ calculated for C₁₉H₁₈ ³⁵ClN₂O₄ 373.0955; found 373.0957.

Reference Compound E:Methyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate

A suspension of P₄S₁₀ (36.1 g, 81.1 mmol) and Na₂CO₃ (8.6 g, 81.1 mmol)in 1,2-DCE (700 mL) at room temperature was stirred for 2 h beforeMethyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-oxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound D) (16.8 g, 45.1 mmol) wasadded. The resulting mixture was stirred at 70° C. for 2 h before beingcooled and filtered. The solid was washed twice with DCM and thefiltrate washed with sat. NaHCO₃ and brine. The organic layer was driedover Na₂SO₄, filtered and concentrated under reduced pressure. The crudeproduct was purified by flash-chromatography on silica gel (DCM/MeOH:99/1) to afford the title compound (17.2 g, 98% yield) as a yellowishsolid. LC/MS (Method A): m/z 389 [M(³⁵Cl)+H]⁺, Rt 2.64 min

HRMS (M+H)⁺ calculated for C₁₉H₁₈ ³⁵ClN₂O₃S 389.0727; found 389.0714.

Reference Compound F:Methyl[(3S)-2-[2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate

To a suspension ofMethyl[(3S)-5-(4-chlorophenyl)-7-(methyloxy)-2-thioxo-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound E (9.0 g, 23.2 mmol) in THF(300 mL) at 0° C. was added hydrazine monohydrate (3.4 mL, 69.6 mmol)dropwise. The reaction mixture was stirred for 5 h between 5° C. and 15°C. before being cooled at 0° C. Et₃N (9.7 mL, 69.6 mmol) was then addedslowly and acetyl chloride (7.95 mL, 69.6 mmol) was added dropwise. Themixture was then allowed to warm to room temperature for 16 h beforebeing concentrated under reduced pressure. The crude product wasdissolved in DCM and washed with water. The organic layer was dried overNa₂SO₄, filtered and concentrated in vacuo to give the crude titlecompound (9.7 g, 98% yield) which was used without further purification.R_(f)=0.49 (DCM/MeOH: 90/10).

Reference Compound G:Methyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate

The crudeMethyl[(3S)-2-[(1Z)-2-acetylhydrazino]-5-(4-chlorophenyl)-7-(methyloxy)-3H-1,4-benzodiazepin-3-yl]acetate(for a preparation see Reference compound F) (assumed 9.7 g) wassuspended in THF (100 ml) and AcOH (60 mL) was added at roomtemperature. The reaction mixture was stirred at this temperature for 2days before being concentrated under reduced pressure. The crude solidwas triturated in i-Pr₂O and filtered to give the title compound (8.7 g,91% over 3 steps) as an off-white solid.

HRMS (M+H)⁺ calculated for C₂₁H₂₀ClN₄O₃ 411.1229; found 411.1245.

Reference Compound H:[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid

To a solution ofMethyl[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetate(for a preparation see Reference compound G) (7.4 g, 18.1 mmol) in THF(130 mL) at room temperature was added 1N NaOH (36.2 mL, 36.2 mmol). Thereaction mixture was stirred at this temperature for 5 h before beingquenched with 1N HCl (36.2 mL) and concentrated in vacuo. Water is thenadded and the aqueous layer was extracted with DCM (×3) and the combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to give the title compound (7 g, 98% yield) as a paleyellow solid.

Reference Compound I:1,1-dimethylethyl[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate

A mixture of[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]aceticacid (for a preparation see Reference compound H) (1.0 g, 2.5 mmol),HATU (1.9 g, 5 mmol) and DIPEA (0.88 ml, 5 mmol) was stirred for 80minutes at room temperature, to this was added 1,1-dimethylethyl(4-aminobutyl)carbamate (1.05 ml, 5.0 mmol, available from Aldrich). Thereaction mixture was stirred at room temperature for 2 h before it wasconcentrated. The residue was taken up in dichloromethane and washedwith 1N HCl. The aqueous layer was extracted with dichloromethane twice.Organic layer was washed with 1N sodium hydroxide, followed by asaturated solution of sodium chloride, dried over sodium sulphate andconcentrated. The residue was purified by flash-chromatography on silicausing dichloromethane/methanol 95/5 to give the title compound as ayellow solid (1.2 g). LC/MS (Method A): rt=3.04 min.

Reference Compound J:N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamidetrifluoroacetate

To a solution of1,1-dimethylethyl[5-({[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetyl}amino)pentyl]carbamate(for a preparation see Reference compound H) (0.2 g, 0.34 mmol) indichloromethane (3 ml) was added trifluoroacetic acid (0.053 ml, 0.68mmol) dropwise at 0° C. The reaction mixture was stirred for 3 h from 0°C. to room temperature. The reaction mixture was concentrated to drynessto afford the title compound as a hygroscopic yellow oil (200mg)

LC/MS (Method A): rt=2.33 min.

HRMS (M+H)⁺ calculated for C₂₅H₂₉ClN₆O₂ 481.2119; found 481.2162.

Reference Compound K: Mixture of 5- and 6-Isomers of Alexa Fluor488-N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamide

N-(5-aminopentyl)-2-[(4S)-6-(4-chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]acetamidetrifluoroacetate (for a preparation see Reference compound J) (7.65 mg,0.013 mmol) was dissolved in N,N-Dimethylformamide (DMF) (300 μl) andadded to Alexa Fluor 488 carboxylic acid succinimidyl ester (5 mg, 7.77μmol, mixture of 5 and 6 isomers, available from Invitrogen, productnumber A-20100) in an Eppendorf centrifuge tube. Hunig's base (7.0 μl,0.040 mmol) was added and the mixture vortex mixed overnight. After 18 hthe reaction mixture was evaporated to dryness and the residueredissolved in DMSO/water (50%, <1 ml total), applied to a preparativePhenomenex Jupiter C18 column and eluted with a gradient of 95% A: 5% Bto 100% B (A=0.1% trifluoroacetic acid in water, B=0.1% TFA/90%acetonitrile/10% water) at a flow rate of 10 ml/min over 150 minutes.Impure fractions were combined and re-purified using the same system.Fractions were combined and evaporated to yield the title product (2.8mg) as a mixture of the 2 regioisomers shown.

LC/MS (Method B):, MH+=999, rt=1.88 min.

Biological Test Methods

Fluorescence Anisotropy Binding Assay

The binding of the compound of formula (I) to Bromodomain 2, 3 and 4 wasassessed using a Fluorescence Anisotropy Binding Assay.

The Bromodomain protein, fluorescent ligand (Reference compound K seeabove) and a variable concentration of test compound are incubatedtogether to reach thermodynamic equilibrium under conditions such thatin the absence of test compound the fluorescent ligand is significantly(>50%) bound and in the presence of a sufficient concentration of apotent inhibitor the anisotropy of the unbound fluorescent ligand ismeasurably different from the bound value.

All data was normalized to the mean of 16 high and 16 low control wellson each plate. A four parameter curve fit of the following form was thenapplied:

y=a+((b−a)/(1+(10̂x/10̂c)̂d)

Where ‘a’ is the minimum, ‘b’ is the Hill slope, ‘c’ is the pIC50 and‘d’ is the maximum.

Recombinant Human Bromodomains (Bromodomain 2 (1-473), Bromodomain 3(1-435) and Bromodomain 4 (1-477)) were expressed in E. coli cells (inpET15b vector) with a six-His tag at the N-terminal. The His-taggedBromodomain was extracted from E. coli cells using 0.1 mg/ml lysozymeand sonication. The Bromodomain was then purified by affinitychromatography on a HisTRAP HP column, eluting with a linear 10-500 mMImidazole gradient, over 20 Cv. Further purification was completed bySuperdex 200 prep grade size exclusion column. Purified protein wasstored at −80 C in 20 mM HEPES pH 7.5 and 100 mM NaCl.

Protocol for Bromodomain 2: All components were dissolved in buffercomposition of 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM CHAPS withfinal concentrations of Bromodomain 2, 75 nM, fluorescent ligand 5 nM.10 μl of this reaction mixture was added using a micro multidrop towells containing 100 nl of various concentrations of test compound orDMSO vehicle (1% final) in Greiner 384 well Black low volume microtitreplate and equilibrated in dark 60 mins at room temperature. Fluorescenceanisotropy was read in Envision (λex=485 nm, λEM=530 nm; Dichroic −505nM).

Protocol for Bromodoamin 3: All components were dissolved in buffer ofcomposition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM CHAPS with finalconcentrations of Bromodomains 3 75 nM, fluorescent ligand 5 nM. 10 μlof this reaction mixture was added using a micro multidrop to wellscontaining 100 nl of various concentrations of test compound or DMSOvehicle (1% final) in Greiner 384 well Black low volume microtitre plateand equilibrated in dark 60 mins at room temperature. Fluorescenceanisotropy was read in Envision (λex=485 nm, λEM=530 nm; Dichroic −505nM).

Protocol for Bromodomain 4: All components were dissolved in buffer ofcomposition 50 mM HEPES pH7.4, 150 mm NaCl and 0.5 mM CHAPS with finalconcentrations of Bromodomain 4 75 nM, fluorescent ligand 5 nM. 10 μl ofthis reaction mixture was added using a micro multidrop to wellscontaining 100 nl of various concentrations of test compound or DMSOvehicle (1% final) in Greiner 384 well Black low volume microtitre plateand equilibrated in dark 60 mins at room temperature. Fluorescenceanisotropy was read in Envision (λex=485 nm, λEM=530 nm; Dichroic −505nM).

Example 1 had a pIC50≧6.0 in each of the BRD2, BRD3 and BRD4 assaysdescribed above.

LPS Stimulated Whole Blood Measuring TNFα Levels Assay

Activation of monocytic cells by agonists of toll-like receptors such asbacterial lipopolysaccharide (LPS) results in production of keyinflammatory mediators including TNFα. Such pathways are widelyconsidered to be central to the pathophysiology of a range ofauto-immune and inflammatory disorders.

Compounds to be tested are diluted to give a range of appropriateconcentrations and 1 ul of the dilution stocks is added to wells of a 96plate. Following addition of whole blood (130 ul) the plates areincubated at 37 degrees (5% CO2) for 30 min before the addition of 10 ulof 2.8 ug/ml LPS, diluted in complete RPMI 1640 (final concentration=200ng/ml), to give a total volume of 140 ul per well. After furtherincubation for 24 hours at 37 degrees, 140 ul of PBS are added to eachwell. The plates are sealed, shaken for 10 minutes and then centrifuged(2500 rpm×10 min). 100 ul of the supernatant are removed and TNFα levelsassayed by immunoassay (typically by MesoScale Discovery technology)either immediately or following storage at −20 degrees. Dose responsecurves for each compound was generated from the data and an IC50 valuewas calculated.

Example 1 was found to have a pIC50>6.0 in the above assay.

These data demonstrate that Example 1 tested in the above assayinhibited the production of the key inflammatory mediator TNFα. Thissuggests that such a compound has a strong anti-inflammatory profile,which is likely to translate into clinical benefit in inflammatorydisorders.

In Vivo Mouse Endotoxemia Model

High doses of Endotoxin (bacterial lipopolysaccharide) administered toanimals produce a profound shock syndrome including a stronginflammatory response, dysregulation of cardiovascular function, organfailure and ultimately mortality. This pattern of response is verysimilar to human sepsis and septic shock, where the body's response to asignificant bacterial infection can be similarly life threatening.

To test the compound of formula (I) and pharmaceutically acceptablesalts thereof groups of eight Balb/c male mice were given a lethal doseof 15 mg/kg LPS by intraperitoneal injection. Ninety minutes later,animals were dosed intravenously with vehicle (20% cyclodextrin 1%ethanol in apyrogen water) or compound (10 mg/kg). The survival ofanimals was monitored at 4 days.

Numbers of animals surviving at 4 days (summed across multiple repeatexperiments)

Vehicle  4/66  (6%) Example 1 24/56 (52%)

These data demonstrate that Example 1 tested in the above model gaverise to a significant animal survival effect following intravenousadministration. This suggests that the compound of formula (I) has thepotential for a profound effect on inflammatory responses in humans.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

1. A compound of formula (I) which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide

or a salt thereof.
 2. A compound of formula (I) which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide

or a pharmaceutically acceptable salt thereof.
 3. A compound of formula(I) which is2-[(4S)-6-(4-Chlorophenyl)-1-methyl-8-(methyloxy)-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepin-4-yl]-N-ethylacetamide


4. A pharmaceutical composition which comprises a compound of formula(I) or a pharmaceutically acceptable salt thereof as defined in claim 2and one or more pharmaceutically acceptable carriers, diluents orexcipients.
 5. A pharmaceutical composition which comprises a compoundof formula (I) as defined in claim 3 and one or more pharmaceuticallyacceptable carriers, diluents or excipients.
 6. A combinationpharmaceutical product comprising a compound of formula (I) or apharmaceutically acceptable salt thereof as defined in claim 2 togetherwith one or more other therapeutically active agents.
 7. A compound offormula (I) or a pharmaceutically acceptable salt thereof as defined inclaim 2 for use in therapy.
 8. A compound of formula (I) as defined inclaim 3 for use in therapy.
 9. A compound of formula (I) or apharmaceutically acceptable salt thereof as defined in claim 2, for usein the treatment of diseases or conditions for which a bromodomaininhibitor is indicated.
 10. A compound of formula (I) or apharmaceutically acceptable salt thereof for use according to claim 9,wherein the disease or condition is a chronic autoimmune and/orinflammatory condition.
 11. A compound of formula (I) or apharmaceutically acceptable salt thereof for use according to claim 9,wherein the disease or condition is cancer.
 12. The use of a compound offormula (I) or a pharmaceutically acceptable salt thereof as defined inclaim 2, in the manufacture of a medicament for the treatment ofdiseases or conditions for which a bromodomain inhibitor is indicated.13. A method for treatment of a disease or condition, for which abromodomain inhibitor is indicated, in a subject in need thereof whichcomprises administering a therapeutically effective amount of compoundof formula (I) or a pharmaceutically acceptable salt thereof as definedin claim
 2. 14. A method for treatment according to claim 13, whereinthe disease or condition is a chronic autoimmune and/or inflammatorycondition.
 15. A method for treatment according to claim 13, wherein thedisease or condition is cancer.
 16. A method for treatment according toclaim 13, wherein the subject is a human.
 17. A method for inhibiting abromodomain which comprises contacting the bromodomain with a compoundof formula (I) or a pharmaceutically acceptable salt thereof as definedin claim 2.